Type 1 diabetes is an autoimmune disease characterized by the progressive destruction of pancreatic beta-cells that leads to insulinopenia and hyperglycemia. In spite of extensive study, little is known about the initial beta-cell autoantigens that trigger the autoimmune response. This application focuses on identifying the antigens that the immune system targets early in the pathogenesis of diabetes. Specific Aim 1: Characterize autoantibodies to beta cell antigens and identify the antigens. We plan on producing monoclonal antibodies from NOD mice of various ages that are reactive with beta cell surface antigens. These antibodies will be characterized and then used to identify the target antigens present on the surface of the beta cells. These beta cell target antigens will be cloned, sequenced and expressed in cell lines by transfection, and in systems that will allow purification of protein for experimentation. Specific Aim 2: Characterize the T cell reactivity against autoantigens that are identified by the monoclonal antibodies. The generation of peptides, and/or protein, from identified beta-cell autoantigens will allow for the pursuit of autoreactive T cells from the spleen, peripancreatic lymph nodes and islet infiltrates of NOD mice. T cell reactivity against all beta cell autoantigens that are identified will be characterized at the level of protein and T cell peptide epitopes will be characterized. Specific Aim 3: Evaluate the pathogenic role of autoantibodies and T cells. The functional role played by any identified beta-cell antigens in the pathogenesis of diabetes, and the antibodies and T cells which target them, will be examined. The phenotype of T cells reactive with identified antigens will be explored and characterized. Together these aims may lead to an understanding of the early pathologic events in Type 1 diabetes, specifically of the beta-cell antigens that are involved, which is essential for the development of preventive and therapeutic interventions.